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L0301P20 - Protein Synthesis
Translation *RNA-directed polypeptide synthesis *eukaryotes: **transcription - nucleus **translation - cytoplasm *requires mRNA, tRNAs, activating enzymes and ribosomes *amino acids are linked together in the codon-specified order of mRNA *achieved by using an adaptor, tRNA, which carries the amino acid and “read” the codon by complementary base pairing Transfer RNA *are small and contain 75-80 nucleotides *have a 3D shape maintained by H bonds arising from internal base pairing *conformation of tRNA facilitates its interaction with components of the protein synthesis machinery *at the top: 3’ end - amino acid always attached to CCA sequence *at the bottom: anticodon **to interact directly with the mRNA by complementary base pairing **provides the contact point **is complementary (and antiparallel) to the mRNA codon tRNA and Wobble *there is NOT one tRNA for every codon *some codon-anticodon interactions can tolerate a mismatch at the 3’ base of the codon (5’ base of the anticodon) *this toleration for the mismatch is known as wobble **e.g. alanine - GCC, GCA, GCU *wobble still does not allow the genetic code to be ambiguous Pre-Translation Preparation *i.e. linking tRNA and amino acids *at the 3’ end of every tRNA is a site to which its specific amino acid is covalently bound *aminoacyl-tRNA synthetases **family of activating enzymes **catalyse linking of amino acid to the tRNA **often results in charging the tRNA **each activating enzyme has a three part active sit that is specific for one amino acid, ATP and its tRNA *bond between tRNA and amino acid provides the energy for making the peptide bond that will join adjacent amino acids Ribosomes *molecular workbench for translation *each ribosome has two subunits: **a large (L) one ***eukaryotes: 3 rRNAs and 45 proteins ***3 sites where tRNA can bind **a small (S) one ***eukaryotes:1 rRNA and 33 proteins *when they are not translating, the two subunits are separate *protein and rRNA components are held together by ionic bonds and dispersion forces *ribosomes are nonspecific and can combine with any mRNA and all tRNAs Binding Sites *tRNA traverses sites in order A—> P—> E **A: amino acid - anticodon binds to codon **P: polypeptide - a.acid added to the chain **E: exit - where the tRNA goes before leaving the ribosome Translation Initiation *begins with an initiation complex **includes the first tRNA and its amino acid (aways methionine - AUG - may be trimmed after synthesis) **an S ribosomal subunit **an mRNA that is to be translated *complex binds to a region towards the 5’ end of the mRNA where the reading begins *initiation complex combines with the large subunit to form the full ribosome *process is directed by initiation factors (proteins) which uses high amounts of GTP Elongation *the L subunit catalyses 2 reactions: **breakage of the bond between the tRNA in the P site and its amino acid (on the polypeptide) **peptide bond formation between this (tRNA-attached) amino acid and the tRNA in the A site *enzyme involved: peptidyl transferase *steps assisted by elongation factors *ribosome moves along the mRNA one codon at a time in the 5’ to 3’ direction *polypeptides grow from the N terminus (amino group) toward the C terminus (carboxyl group) Process #first tRNA enter the open A, moves to P, opening the A for next tRNA #first tRNA releases methionine, dissociates from ribosome, open P #2nd tRNA then moves to P (opens A) #next charged tRNA enters the open A #the peptide chain is transferred to the tRNA occupying P site Termination *when stop codon (UAA, UAG or UGA) reaches A site, release factor enters A site instead of an amino acid *causes translation to terminate and newly completed polypeptide to separate from the ribosome *subunits also separate *mRNA and release factor are released Antibacterial Antibiotics *antibiotics have been used to combat human bacterial infectious disease *must specifically destroy microbial invaders, but not harm the human host *some antibiotics work by blocking events in translation *due to difference between prokaryotic and eukaryotic ribosome proteins, human ribosomes are unaffected Regulation of Translation *nothing stops more than one ribosome working on the one mRNA at one time *in a poly(ribo)some, more than one ribosome moves along the mRNA at one time *allows simultaneous translation of the mRNA by multiple ribosomes Post-Translational Events *newly synthesised polypeptide have signals (“address labels”) contained in the amino acid sequences that direct them to a particular cellular destination Protein synthesis begins on free ribosomes in the cytoplasm however has two end outcomes: Option 1: Finish and Release to Organelles *proteins destined for nucleus, mitochondria, peroxisomes etc are completed there *have signals that allow them to bind to and enter the destined organelles Option 2: Stall translation and go to ER *these are proteins destined for the ER, Golgi, lysosomes, plasma membrane and outside the cell wall *enter ER by interaction of hydrophobic signal sequence with a channel in the membrane *embedding into the membrane: **have ‘anchor’ sequences which ensure their retention in membranes **membrane proteins that get to the plasma membrane are orientated to face the outside of the cell *covalent modifications of proteins of proteins occur in the ER and can be modified in the GA *proteins with no signals go from the ER through the GA and are secreted from the cell (in vesicles) via exocytosis Protein Modification *as polypeptide chain forms, it folds into its 3D shape: **some occur spontaneously **some required help proteins - chaperones *covalent modifications of proteins after translation may include: **proteolysis (cleavage of the protein) ***insulin **glycosylation (addition of sugars) **phosphorylation (addition of phosphate groups) *contribute to shape and interactions and are essential for the final function of the protein